title: One-step enzyme extraction and immobilization for biocatalysis applications.
abstract: An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.
authors.affiliation: Division of Biochemistry, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
authors.forename: Karim Engelmark
authors.initials: KE
authors.lastname: Cassimjee
authors.forename: Robert
authors.initials: R
authors.lastname: Kourist
authors.forename: Diana
authors.initials: D
authors.lastname: Lindberg
authors.forename: Marianne
authors.initials: M
authors.lastname: Wittrup Larsen
authors.forename: Nguyen Hong
authors.initials: NH
authors.lastname: Thanh
authors.forename: Mikael
authors.initials: M
authors.lastname: Widersten
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
authors.forename: Per
authors.initials: P
authors.lastname: Berglund
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000302:isolation & purification*; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000302:isolation & purification; D005656:Fungal Proteins; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D010940:Plant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica; D004851:Epoxide Hydrolases